Expression and production of polypeptides using the promoters of the hansenula polymorpha MOX and DAS genes

ABSTRACT

The structural genes and their regulatory DNA sequences of an alcohol oxidase (MOX) and a dihydroxyacetone synthase (DHAS) of Hansenula polymorpha have been isolated and the nucleotide sequences determined. The invention relates to the use of the MOX gene, as well as the use of the regulatory DNA sequences of MOX and/or DAS in combination with the MOX gene, optionally after modification thereof, or other oxidase genes, or other genes, to produce engineered microorganisms, 0in particular yeasts. Said engineered microorganisms can produce oxidases or other enzymes in yields that allow industrial application on a large scale. Moreover, said engineered microorganisms can produce oxidases having improved properties with respect to their application in oxidation reactions and/or in bleaching and detergent products.

This a continuation of appln. Ser. No. 08/045,081, filed on Apr. 12,1993, which was abandoned upon the filing hereof, which was acontinuation of appln. Ser. No. 07/587,555 filed Sep. 24, 1990, now U.S.Pat. No. 5,340,838 which was continuation of appln. Ser. No. 07/300,211filed Jan. 23, 1989, now abandoned, which is a continuation of appln.No. 06/759,315, filed Jul. 26, 1995, now abandoned.

The present invention relates to a process for microbiologicallypreparing oxidoreductases, use of these enzymes in bleaching and/ordetergent compositions, as well as to microorganisms transformed by DNAsequences coding for an oxidoreductase and optionally for adihydroxyacetone synthase-enzyme, and H. polymorpha alcohol oxidaseand/or dihydroxyacetone synthase regulation sequences, themicroorganisms being suitable for use in the process.

Oxidoreductases, especially those which use oxygen as electron acceptor,are enzymes suitable for use in bleaching and/or detergent compositionsin which they can be used for the in situ formation of bleaching agents,e. g. H₂ O₂, during the washing or bleaching process. See for example

GB-PS 1 225 713 (Colgate-Palmolive Company), in which the use of amixture of glucose and glucose oxidase and other ingredients in a drypowdered detergent composition has been described,

DE-PA 2 557 623 (Henkel & Cie GmbH), in which the use of a C₁ to C₃alkanol and alcohol oxidase, or galactose and galactose-oxidase, or uricacid and uratoxidase, and other ingredients in a dry detergentcomposition having bleaching properties has been described, and

GB-PA 2 101 167 (Unilever PLC) in which the use of a C₁ to C₄ alkanoland a C₁ to C₄ alkanol oxidase in a liquid bleach and/or detergentcomposition has been described,

wherein the alkanol and the enzyme are incapable of substantialinteraction until the composition is diluted with water, and/or has comeinto contact with sufficient oxygen.

Up to now natural oxidase-enzymes cannot be produced at a cost pricethat allows industrial application on a large scale, e.g. detergentproducts. Moreover, the oxidase-enzymes have to act undernon-physiological conditions when used in detergent and bleachingproducts. Further the natural oxidases that have been investigated foruse in detergent compositions are accompanied by the naturalcatalase-enzyme which decomposes almost immediately the peroxide(s)formed, so that no effective bleaching is obtained. Thus a need existsfor oxidase-enzymes that are more suitable for use under the conditionsof manufacture and use of detergent and bleaching products.

For an economically feasible production of these oxidases it is furtherrequired to reach a yield of these enzymes in fermentation processes inthe order of that of alcohol oxidase of H. polymorpha, which is up to20% of the cellular protein (van Dijken et al., 1976).

One way of finding new microorganisms producing enzymes in higheramounts or finding new oxidase-enzymes having improved properties is tocheck all sorts of microorganisms and try to isolate the relevantoxidases, which are then checked for their abilities to generateperoxides and their stabilities under the conditions of manufacture anduse of detergent and bleaching products. One can hope that some day asuitable enzyme will be found, but the chance of success isunpredictable and probably very low.

Another way is to apply another trial and error method of crossing thenatural microorganisms producing these oxidases by classical genetictechniques, in the hope that some day one will find a more productivemicroorganism or a more suitable enzyme, but again the chance of successis rather low.

Clearly, a need exists for a method for preparing oxidase-enzymes inhigher yield and/or without the concomitant formation of catalase and/orhaving improved properties during storage and/or use in e.g. bleachand/or detergent compositions. The problem of trial and error can beovercome by a process for preparing an oxidase-enzyme by culturing amicroorganism under suitable conditions, and preferably concentratingthe enzyme and collecting the concentrated enzyme in a manner known perse, which process is characterized in that a microorganism is used thathas been obtained by recombinant DNA technology and which is capable ofproducing said oxidase-enzyme.

The microorganisms suitable for use in a process for preparing anoxidase-enzyme can be obtained by recombinant DNA technology, whereby amicroorganism is transformed by a DNA sequence coding for anoxidase-enzyme (so-called structural gene) together with one or moreother DNA sequences which regulate the expression of the structural genein a particular microorganism or group of organisms, either viaintroduction of an episomal vector containing said sequences or via avector containing said sequences which is also equipped with DNAsequences capable of being integrated into the chromosome of themicroorganism.

The determination of a structural gene coding for the enzyme alcoholoxidase (EC 1.1.3.13) originating from H. polymorpha together with itsregulatory 5'- and 3'-flanking regions will be described as an exampleof the invention without the scope of the invention being limited tothis example. The spirit of the invention is also applicable to theisolation of DNA sequences of other oxidase-enzymes such as glyceroloxidase, glucose oxidase, D-amino acid oxidase etc.; the incorporationof the DNA sequences or modifications thereof into the genome ofmicroorganisms or into episomal vectors used for transformingmicroorganisms and the culturing of the transformed microorganisms soobtained as such or for producing the desired oxidase-enzymes, as wellas the use of these enzymes in bleaching compositions containing them.

Although the microorganisms to be used can be bacteria, e.g. of thegenus Bacillus, as well as moulds, the use of yeasts is preferred fortechnological and economical reasons. In particular a mould or yeast canbe selected from the genera Aspergillus, Candida, Geotrichum, Hansenula,Lenzites, Nadsonia, Pichia, Poria, Polyporus, Saccharomyces,Sporobolomyces, Torulopsis, Trichosporon and Zendera, more particularlyfrom the species A. japonicus, A. niger, A. oryzae, C. boidinii, H.polymorpha, Pichia pastoris and Kloeckera sp. 2201. The latter name issometimes used instead of C. boidinii.

Many C₁ -utilizing yeasts have been isolated during the last decade, andfor Hansenula polymorpha and Candida boidinii the methanol metabolismhas been studied extensively (for a review see Veenhuis et al., 1983).

The first step in this metabolism is the oxidation of methanol toformaldehyde and H₂ O₂ catalysed by MOX. Formaldehyde is oxidizedfurther by the action of formaldehyde dehydrogenase and formatedehydrogenase. H₂ O₂ is split into water and oxygen by catalase.

Alternatively, methanol is assimilated into cellular material. After itsconversion into formaldehyde, this product is fixed through the xylulosemonophosphate pathway into carbohydrates. Dihydroxyacetone synthase(DHAS) plays a crucial role in this assimilation process.

The appearance of MOX, formate dehydrogenase, formaldehydedehydrogenase, DHAS and catalase is subject to glucose repression, e.g.on 0.5% glucose. However, synthesis of MOX is derepressed by growth inlow concentrations of glucose (0.1%), contrary to the synthesis of DHAS,which is still fully repressed under these conditions (Roggenkamp etal., 1984).

Regulation, i.e. the possibility to switch "on" or "off" of the gene forthe polypeptide concerned, is desirable, because it allows for biomassproduction, when desired, by selecting a suitable substrate, such as,for example melasse, and for production of the polypeptide concerned,when desired, by using methanol or mixtures of methanol and other carbonsources. Methanol is a rather cheap substrate, so the polypeptideproduction may be carried out in a very economical way.

After derepression of the gene coding for alcohol oxidase (MOX) bygrowth on methanol, large microbodies, the peroxisomes are formed. Whileglucose-grown cells contain only a small peroxisome, up to 80% of theinternal volume of the cell is replaced by peroxisomes in thederepressed state. The conversion of methanol into formaldehyde and H₂O₂ as well as the degradation of H₂ O₂ has been shown to occur in theseperoxisomes, while further oxidation or assimilation of formaldehydemost probably occurs in the cytoplasm. This process is a perfect exampleof compartmentalization of toxic products, of a strong co-ordinatederepression of several cellular processes and of the selectivetranslocation of at least two of the enzymes involved in this process.

Most of the enzymes involved in the methanol metabolism have beenpurified and characterized (Sahm, 1977, Bystrykh et al, 1981).Especially methanol oxidase (EC 1.1.3.13) has been studied in detail. Itis an octamer consisting of identical monomers with an M_(r) value ofabout 74 kd and it contains FAD as a prosthetic group. Up to now nocleavable signal sequence for translocation could be detected, asconcluded from electroelephoresis studies with in vivo and in vitrosynthesized products (Roa and Blobel, 1983) or from in vitro synthesisin the presence of microsomal membranes (Roggenkamp et al., 1984).

Under derepressed conditions, up to 20% of the cellular protein consistsof MOX.

Materials and methods

a) Microorganisms and cultivation conditions

Hansenula polymorpha CBS 4732 was obtained from Dr J. P. van Dijken(University of Technology, Delft, The Netherlands). Cells were grown at37° C. in 1 liter Erlenmeyer flasks containing 300 ml minimal medium(Veenhuis et al., 1978), supplemented with 0.5% (v/v) methanol or 0.5%(v/v) ethanol as indicated. Phage lambda L47.1 and the P2 lysogenic E.coli K12 strain Q 364 were obtained from Dr P. van der Elsen (FreeUniversity of Amsterdam, The Netherlands) and propagated as described(Loenen and Brammar, 1980).

E. coli K12 strains BHB 2600, BHB 2688 and BHB 2690 (Hohn, 1979) wereobtained from Dr M. van Montagu (University of Gent, Belgium), while E.coli K12 strain JM 101.7118 and the M13 derivatives M13 mp 8, 9, 18 and19 were obtained from Bethesda Research Laboratories Inc. (Gaithersburg,Md., U.S.A.).

b) Enzymes

All enzymes used were obtained from Amersham International PLC,Amersham, U.K., except alpha-helicase which was obtained from PharmIndustrie, Clichy, France. Enzyme incubations were performed accordingto the instructions of the manufacturer. ATP:RNA adenyl transferase waspurified as described by Edens et al. (1982).

c) Other materials

³⁵ S! methionine, alpha-³⁵ S! dATP, alpha-³² P! dNTP's, alpha-³² P! ATPand gamma-³² P! ATP were obtained from Amersham International PLC,Amersham, U.K.

Nitrobenzyloxy-methyl (NBM) paper was obtained from Schleicher andSchuell, and converted into the diazo form (DBM) according to theinstructions of the manufacturer.

Nitrocellulose filters (type HATF) were obtained from Millipore.

RNA isolation, fractionation and analysis

Hansenula polymorpha cells were grown to mid-exponential phase, eitherin the presence of methanol or ethanol. The cells were disrupted byforcing them repeatedly through a French Press at 16 000 psi, in abuffer containing 10 mM Tris-HCl pH 8, 5 mM MgCl₂, 1% NaCl, 6%para-aminosalicylic acid, 1% sodium dodecylsulphate (SDS) and 5% phenol.The purification of polyadenylated RNA was subsequently performed, asdescribed previously (Edens et al., 1982). One gram cells yielded fourmg total RNA and 0.1 mg polyadenylated RNA. Five microgram samples oftotal RNA or polyadenylated RNA were radioactively labelled at their3'-ends with ATP:RNA adenyl transferase and alpha-³² P! ATP, andsubsequently separated on a 2.5% polyacrylamide gel containing 7M urea(Edens et al., 1982). For the preparative isolation of a specific mRNAfraction, 40 micrograms polyadenylated RNA was mixed with fourmicrograms of labelled polyadenylated RNA and separated on thedenaturing polyacrylamide gel. The radioactive 2.4 kb RNA class waseluted from slices of the gel and freed from impurities bycentrifugation through a 5-30% glycerol gradient in 100 mM NaCl, 10 mMTris-HCl pH 7.5, 1 mM EDTA and 0.1% SDS for 15 h at 24 000 rev./min. ina Beckmann centrifuge using an SW 60 rotor at 20° C. The radioactivefractions were pooled and precipitated with ethanol. Polyadenylated RNAwas translated in vitro in a rabbit reticulocyte lysate according toPelham and Jackson (1976), using ³⁵ S! methionine as a precursor. Thetranslation products were immuno-precipitated with MOX antiserum asdescribed by Valerio et al. (1983).

cDNA synthesis

One third of the RNA fraction, isolated from the polyacrylamide gel, wasused to procure a radioactive cDNA with reverse transcriptase (Edens etal., 1982). Using alpha-³² P! dATP and alpha-³² P! dCTP of a highspecific activity (more than 3000 Ci/mM), 20 000 cpm of high molecularweight cDNA was formed during 1 h at 42° C. in the presence of humanplacental ribonuclease inhibitor.

DNA isolation

Ten g of Hansenula polymorpha cells were washed with 1M sorbitol andresuspended in 100 ml 1.2M sorbitol, 10 mM EDTA and 100 mM citric acidpH 5.8, to which 100 microliter beta-mercapto-ethanol was added. Cellswere spheroplasted by incubation with 500 mg alpha-helicase for 1 h at30° C. Spheroplasts were collected by centrifugation at 4000 rev./min.in a Sorvall GSA rotor, resuspended in 40 ml 20 mM Tris-HCl pH 8, 50 mMEDTA and lysed by adding 2.5% SDS. Incompletely lysed cells werepelleted for 30 min. at 20 000 rev./min. in a Sorvall SS34 rotor and DNAwas isolated from the viscous supernatant by centrifugation using aCsCl-ethidium bromide density gradient at 35 000 rev./min. for 48 h in aBeckmann centrifuge using a 60 Ti rotor. 2 mg of DNA was isolated with amean length of 30 kb.

Preparation of a clone bank in phage lambda L47.1

150 microgram Hansenula polymorpha DNA was partially digested withSau3AI and sedimented through a 10-40% sucrose gradient in 1M NaCl, 20mM Tris-HCl pH 8 and 5 mM EDTA for 22 h at 23 000 rev./min. in an SW 25rotor. The gradient was fractionated and samples of the fractions wereseparated on a 0.6% agarose gel in TBE buffer (89 mM Tris, 89 mM Boricacid, 2.5 mM EDTA).

Fractions that contained DNA of 5-20 kb were pooled and the DNA wasprecipitated with ethanol. Phage lambda L47.1 was grown, and its DNA wasisolated as described by Ledeboer et al. (1984). The DNA was digestedwith BamHI and arms were isolated by centrifugation through a potassiumacetate gradient as described by Maniatis et al. (1982). Two microgramphage lambda DNA arms and 0.5 μg Sau3AI digested Hansenula polymorphaDNA thus obtained were ligated and packaged in vitro using a protocolfrom Hohn (1979). Phages were plated on E. coli strain Q 364 to a plaquedensity of 20,000 pfu per 14 cm Petri dish. Plaques were blotted onto anitrocellulose filter (Benton and Davis, 1977) and the blot washybridized with the radioactive cDNA probe isolated as described above.Hybridization conditions were the same as described by Ledeboer et al.(1984) and hybridizing plaques were detected by autoradiography.

Isolation and partial amino acid sequence analysis of alcohol oxidase(MOX)

Hansenula polymorpha cells grown on methanol were disintegrated byultrasonification and the cell debris was removed by centrifugation. TheMOX-containing protein fraction was isolated by (NH₄)₂ SO₄ precipitation(40-60% saturation). After dialysis of the precipitate, MOX wasseparated from catalase and other proteins by ion-exchangechromatography (DEAE-Sepharose) and gel filtration (Sephacryl S-400).Antibodies against MOX were raised in rabbits by conventional methodsusing complete and incomplete Freund's adjuvants (Difco Lab, Detroit,U.S.A.). Sequence analysis of alcohol oxidase treated with performicacid was performed on a Beckman sequenator. Identification of theresidues was done with HPLC. The amino acid composition was determinedon a Chromaspek analyser (Rank Hilger, U.K.), using standard proceduresand staining by ninhydrine. The carboxy terminal amino acid wasdetermined as described by Ambler (1972).

Chemical synthesis of deoxyoligonucleotides

Deoxyoligonucleotides were synthesized on a Biosearch SAM I genemachine, using the phosphite technique (Matteucci and Caruthers, 1981).They were purified on 16% or 20% polyacrylamide gels in TBE.

Hybridization with deoxyoligonucleotide probes

The deoxyoligonucleotides were radioactively labelled with T₄-polynucleotide kinase and gamma-³² P! ATP. The DNA of the MOX clonesobtained was digested with different restriction enzymes, separated on1% agarose gel and blotted onto DBM paper. Hybridizations were performedas described by Wallace et al. (1981).

DNA sequence analysis

From clone 4 (see Example 1) containing the complete MOX gene, severalsubclones were made in phage M13mp-8, -9 or M13mp-18, -19 derivatives bystandard techniques. Small subclones (less than 0.5 kb), cloned in twoorientations, were sequenced directly from both sides. From the largersubclones, also cloned in two orientations, sequence data were obtainedby an exonuclease Bal31 digestion strategy (see FIG. 1). For each ofboth cloned orientations the RF M13 DNA is digested with a restrictionenzyme that preferably cleaves only in the middle of the insert.Subsequently, both orientations of the clones were cut at this uniquesite, and digested with exonuclease Bal31 at different time intervals.Incubation times and conditions were chosen such that about 100-150nucleotides were eliminated during each time interval. Each fraction wasdigested subsequently with the restriction enzyme, recognizing therestriction site situated near the position at which the sequencereaction is primed in the M13 derivatives. Ends were made blunt end byincubation with T₄ -polymerase and all dNTP's, and the whole mix wasligated under diluted conditions, thereby favouring the formation ofinternal RF molecules. The whole ligation mix was used to transform toE. coli strain JM 101-7118. From each time interval several plaques werepicked up and sequenced using recently described modifications of theSanger sequencing protocol (Biggin et al., 1983).

The isolation of auxotrophic mutants

LEU-1 (CBS N° 7171) is an auxotrophic derivative of H. polymorpha strainNCYC 495 lacking β-isopropylmalate dehydrogenase activity. The isolationof this mutant has been described by Gleeson et al. (1984).

LR9 (CBS N° 7172) is an auxotrophic derivative of H. polymorpha ATCC34438, lacking orotidine 5'-decarboxylase activity.

For the isolation, all procedures were carried out at 30° C. instead of37° C., which is the optimal temperature for growth of this yeast. Yeastcells were mutagenized with 3% ethylmethanesulphonate for 2 hr (Fink,1970). The reaction was stopped with 6% sodium thiosulphate (finalconcentration) and the solution was incubated for another 10 min.Mutagenized cells were then washed once with H₂ O and incubated for 2days on YEPD or YNB supplemented with uracil for segregation andenrichment of uracil-auxotrophs followed by a 15 hr cultivation on MMwithout nitrogen source. Finally a nystatin enrichment was employed for12 hr on MM with a concentration of 10 μg antibiotic per ml. The treatedcells were plated on YNB plates containing 200 μg uracil per ml and 0.8mg 5-fluoroorotic acid (Boeke et al., 1984). Usually 10⁶ cells wereplated on a single plate. Resistant colonies were picked after 3 days ofincubation, replica plated twice on YNB plates to establish theauxotrophy. From the auxotrophic mutants ura⁻ cells were isolated.Alternatively, 1.5×10⁶ yeast cells were incubated in one ml of YNBliquid medium supplemented with 200 μg of uracil and 0.8 mg of5-fluoroorotic acid. After incubation of 2 days, the treated cells wereplated on YNB containing uracil, replica-plated twice on YNB andanalysed as described above.

Such resistant mutants have been shown to be uracil auxotrophs affectedat the URA3 or the URA5 locus in S. cerevisiae (F. Lacroute, personalcommunication). Of about 600 resistant colonies of H. polymorpha tested,52 exhibited a uracil phenotype. Since URA3 and URA5 mutations in S.cerevisiae lack orotidine 5'-decarboxylase and orotidine 5'-phosphatepyrophosphorylase, respectively (Jones and Fink, 1982), the obtaineduracil auxotrophs of H. polymorpha were tested for both enzymaticactivities (Lieberman et al., 1955). Mutants affected in either of thetwo enzymes were found (Table I). They have been designated odcl andoppl mutants, respectively. The odcl mutants exhibit adequate lowreversion frequencies (Table II) and thus are suitable fortransformation purposes by complementation.

Isolation of autonomous replication sequences (HARS) from H. polymorpha

Chromosomal DNA from H. polymorpha was partially digested either withSalI or BamHI and ligated into the single SalI and BamHI site of theintegrative plasmid YIp5, respectively. The ligation mixture was used totransform E. coli 490 to ampicillin resistance. YIp5 is an integrativeplasmid containing the URA3 gene as a selective marker (Stinchcomb etal., 1980).

The plasmid pool of H. polymorpha SalI clones was used to transform H.polymorpha mutant LR9. A total of 27 transformants was obtained beingalso positive in the β-lactamase assay. From all of them, plasmids couldbe recovered after transformation of E. coli 490 with yeast minilysates.Restriction analysis of the plasmids revealed that most of the insertsshow the same pattern. The two different plasmids, pHARS1 and pHARS2,containing inserts of 0.4 and 1.6 kb respectively, were used for furtherstudies (FIG. 2). Both plasmids transform H. polymorpha mutant LR9 witha frequency of about 500-1,500 transformants per μg of DNA using thetransformation procedure of intact cells treated withpolyethyleneglycol. Southern analysis of the H. polymorpha transformantsafter retransformation with pHARS1 and pHARS2 recovered from E. coliplasmid preparations shows the expected plasmid bands and thus excludesintegration of the URA3 gene as a cause of the uracil protrophy.Therefore, we conclude that the HARS sequences like ARS1 (Stinchcomb etal., 1982) allow autonomous replication in H. polymorpha. Neither HARS1nor HARS2 enabled autonomous replication in S. cerevisiae. HARS1 wassequenced completely.

Estimation of plasmid copy number in H. polymorpha transformants

The copy number of plasmids conferring autonomous replication in H.polymorpha either by ARS sequences or by HARS sequences was estimated bySouthern blot analysis (FIG. 3). For comparison, plasmid YRP17 in S.cerevisiae (FIG. 3, lanes 6, 7), which has a copy number of 5-10 percell (Struhl et al., 1979) and the high copy number plasmid pRB58 in S.cerevisiae (FIG. 3, lanes 4, 5) with abut 30-50 copies per cell wereused. YRP17 is a URA3-containing yeast plasmid, bearing an ARS sequence(Stinchcomb et al., 1982), while pRB58 is a 2 μm derivative containingthe URA3 gene (Carlson and Botstein, 1982). A Kluyveromyces lactistransformant carrying 2 integrated copies of pBR pBR322 was used as acontrol (FIG. 3, lanes 2, 3). The intensity of staining in theautoradiogram reveals that the plasmid YRP17 in H. polymorpha haspractically the same copy number as in S. cerevisiae, whereas plasmidspHARS-1 and pHARS-2 show a copy number which is in the range of about30-40 copies per cell like pBR58 in S. cerevisiae. This proves once morethe autonomously replicating character of the HARS sequence.

Transformation procedures

Several protocols were used.

a) H. polymorpha strain LEU-1 was transformed using a procedure adaptedfrom Beggs (1978). The strain was grown at 37° C. with vigorous aerationin 500 ml YEPD liquid medium up to an OD₆₀₀ of 0.5. The cells wereharvested, washed with 20 ml distilled water and resuspended in 20 ml1.2M sorbitol, 25 mM EDTA pH 8.0, 150 mM DTT and incubated at roomtemperature for 15 minutes. Cells were collected by centrifugation andtaken up in 20 ml 1.2M sorbitol, 0.01M EDTA, 0.1M sodium citrate pH 5.8and 2% v/v beta-glucuronidase solution (Sigma 1500000 units/ml) andincubated at 37° C. for 105 minutes. After 1 hr, the final concentrationof beta-glucuronidase was brought to 4% v/v. For transformation, 3 mlaliquots of the protoplasts were added to 7 ml of ice cold 1.2Msorbitol, 10 mM Tris-HCl pH 7. Protoplasts were harvested bycentrifugation at 2000 rpm for 5 minutes and washed three times in icecold sorbitol buffer. Washed cells were resuspended in 0.2 ml 1.2Msorbitol, 10 mM CaCl₂, 10 mM Tris-HCl pH 7 on ice. 2 μg of YEP13 DNA--anautonomous replicating S. cerevisiae plasmid consisting of the LEU2 geneof S. cerevisiae and the 2 micron-ori (Broach et al., 1979)--were addedto 100 ml of cells and incubated at room temperature. 0.5 ml of asolution of 20% PEG 4000 in 10 mM CaCl₂, 10 mM Tris-HCl pH 7.5 was addedand the whole mixture was incubated for 2 minutes at room temperature.Cells were collected by brief (5 sec.) centrifugation in an MSGmicrofuge set at high speed and resuspended in 0.1 ml YEPD 1.2M sorbitolpH 7.0, and incubated for 15 minutes at room temperature. The cells wereplated directly by surface spreading on plates containing 2% Difco agar,2% glucose, 0.67% Difco yeast nitrogen base and 20 mg/1 of each ofL-adenine Hemisulphate, methionine, uracil, histidine, tryptophan,lysine and 1.2M sorbitol. Leu⁺ transformants appear after 5 daysincubation at 37° C. with a frequency of 50 colonies/μg DNA, while notransformants appear if no DNA is added.

b) Alternatively, H. polymorpha LEU-1 was transformed with YEP13, usinga procedure adapted from Das et al. (1984). Exponentially growing cellswere grown up to an OD₆₀₀ of 0.4, washed in TE buffer (50 mM Tris-HCl pH8.0, 1 mM EDTA) and resuspended in 20 ml TE buffer. 0.5 ml cells wereincubated with 0.5 ml 0.2M LiCl for 1 hr at 30° C. To 100 ml of thesecells 4 μg YEP13 in 20 ml TE buffer was added and the sample wasincubated for a further 30 minutes at 30° C. An equal volume of 70% v/vPEG 4000 was added and the mixture was incubated for 1 hr at 30° C.,followed by 5 min. at 42° C. After addition of 1 ml H₂ O, cells werecollected by a brief centrifugation as described under a), washed twicewith H₂ O and resuspended in 0.1 ml YEPD 1.2M sorbitol and incubated for15 minutes at room temperature. Cells were plated as described. Leu⁺transformants appear with a frequency of 30 μg DNA.

c) The H. polymorpha URA mutant LR9 was transformed with YRP17, aplasmid containing the URA3 gene of S. cerevisiae as a selective markerand an autonomously replicating sequence (ARS) for S. cerevisiae(Stinchomb et al, 1982). Using the protoplast method, described by Beggs(1978), 2-5 transformants/μg DNA were obtained. This number wasenlarged, using the LiSO₄ method of Ito et al. (1983), up to 15-20transformants per μg of DNA. However, the best procedure was theprocedure described by Klebe et al. (1983), using intact cells treatedwith PEG 4000. Up to 300 transformants were obtained per μg DNA. TheLiSO₄ procedure, as well as the Klebe procedure, was performed at 37° C.

Transformation of H. polymorpha based on autonomous replication of thevector was indicated by two characteristics: (1) the instability of theuracil⁺ phenotype. After growth of transformants on YEPD for tengenerations, more than 99% had lost the ability to grow on selectivemedium (Table II). (2) Autonomous replication was further ascertained bytransforming E. coli cells with yeast minilysates and retransformationof H. polymorpha. Subsequent Southern analysis showed the presence ofthe expected plasmid.

H. polymorpha LR9 could not be transformed with pRB58, or with pHH85,constructed by insertion of the whole 2 micron circle DNA (Hollenberg,1982) into the PstI site of the ampicillin gene of plasmid YIP5. YIP5,containing the DNA sequence of HARS1 or HARS2, was transferred to H.polymorpha LR9 using the Klebe protocol with a frequency of 500-1500transformants per μg of DNA. Thus, transformation frequency is 2-5 timeshigher than described above, using the heterologous ARS 1 in YRP17 of S.cerevisiae. Similarly, the stability of the HARS plasmid intransformants is slightly higher than the ARS 1 plasmid (Table II).

Transformation of H. polymorpha by integration of the URA3 gene from S.cerevisiae

The URA3 gene of S. cerevisiae shows no homology to the ODC gene in H.polymorpha, as revealed by Southern hybridisation of nick-translatedYIp5 plasmid DNA to chromosomal DNA of H. polymorpha. Therefore,low-frequency integration of the URA3 gene at random sites of the H.polymorpha genome had to be anticipated. Transformation of mutant LR9with the integrative vector YIp5 resulted in 30-40 colonies per μg ofDNA on YNB plates using the polyethyleneglycol method, whereas notransformants were obtained in the control experiment using YIp5 fortransformation of S. cerevisiae mutant YNN27. Analysis of 38transformants revealed 4 stable integrants after growth on non-selectivemedium. The integration event was further demonstrated by Southernanalysis (FIG. 4).

A second procedure for generating integration of the URA3 gene intochromosomal DNA of H. polymorpha was performed by enrichment of stableUra⁺ transformants from transformants carrying plasmid pHARS1.Transformants were grown in liquid YEPD up to a density of 10⁹ cells perml. An aliquot containing 5×10⁶ cells was used to inoculate 100 ml offresh medium and was grown up to a cell density of 10⁹ per ml. Theprocedure was repeated until about 100 generations had been reached.Since the reversion rate,of mutant LR9 is 2×10⁻⁹ and the frequency ofplasmid loss per 10 generations is 97% in pHARS1 transformants, thepredominant part of the Ura⁺ cells after 100 generations should beintegrants. The Ura⁺ colonies tested were all shown to maintain a stableUra⁺ phenotype indicating an integration of the URA3 gene. This wasfurther verified by Southern blot analysis. In addition, these dataindicate that the integration frequency is 5×10⁻⁶.

EXAMPLE 1 CLONING OF THE GENE FOR ALCOHOL OXIDASE (MOX) FROM HANSENULAPOLYMORPHA

Characterization of polyadenylated RNA

Total RNA and polyadenylated RNA, isolated from cells grown on methanol,were labelled at their 3'-termini with ATP:RNA adenyl transferase, andseparated on a denaturing polyacrylamide gel (FIG. 5). Apart from therRNA bands, two classes of RNA appear in the poly-adenylated RNA lane,respectively 1 kb and 2.3 kb in length. Since these RNA classes are notfound in polyadenylated RNA of ethanol-grown cells (result not shown),they obviously are transcripts of genes derepressed by growth onmethanol. The 2.3 kb class can code for a protein of 700 to 800 aminoacids, depending on the length of the non-translated sequences.Likewise, the 1 kb class codes for a protein of 250-300 amino acids.Enzymes that are derepressed by growth on methanol and are 700 to 800amino acids long, most likely are MOX (Kato et al., 1976; Roa andBlobel, 1983) and DHAS (Bystrykh et al., 1981). Derepressed enzymes inthe 250 to 300 amino acid range are probably formaldehyde and formatedehydrogenase (Schutte et al., 1976). The polyadenylated RNA wascharacterized further by in vitro translation in a reticulocyte cellfree translation system. Two microliters of the polyadenylated RNAdirected protein mixture were separated directly on a 10% SDSpolyacrylamide gel, while the remaining 18 microliters were subjected toimmuno-precipitation with antiserum against MOX (FIG. 6). Six strongbands dominate in the total protein mixture, having molecular weights ofrespectively 78 kd, 74 kd, 58 kd, 42 kd, 39 kd and 36 kd. Essentiallythe same molecular weights were found by

Roa and Blobel (1983) in a total cell extract from methanol-grown H.polymorpha cells.

The 74 kd protein can tentatively be assigned to the monomer of MOX, the58 kd protein to the monomer of catalase and the 39 kd and 36 kdproteins to the monomers of formaldehyde dehydrogenase and formatedehydrogenase, respectively. The 78 kd polypeptide possibly is DHAS,while the 42 kd polypeptide remains unidentified. Afterimmuno-precipitation, both high molecular weight proteins react with theMOX antiserum.

Cloning of the gene for MOX

Although the 2.3 kb mRNA class induced by growth on methanol obviouslycodes for at least 2 polypeptides, it seemed a good candidate forscreening a Hansenula polymorpha clone bank by hybridization. The 5-20kb fraction of partially Sau3AI digested H. polymorpha DNA was cloned inphage lambda L47.1.

Per microgram insert DNA, 300 000 plaques were obtained while thebackground was less than 1:1000. Two Benton Davis blots, containingabout 20 000 plaques each, were hybridized with 15 000 cpm of themRNA-derived cDNA probe. After 3 weeks of autoradiography about 40-50hybridizing plaques could be detected. All plaques were picked up andfive were purified further by plating at lower density and by a secondhybridization with the cDNA probe. From four, single hybridizing plaques(1, 3, 4, 5) DNA was isolated. The insert length varied from 8 to 13 kb.

Hybridization selection using organic-synthetic DNA probes

The sequence of 30 amino acids at the amino terminus of purified MOX wasdetermined (FIG. 7).

Using the most abundant codon use for the yeast S. cerevisiae, asequence of 14 bases could be derived from part of this proteinsequence, with only one ambiguity. Both probes, indicated in FIG. 3,were synthesised. In both probes an EcoRI site is present. DBM blotswere made from the DNA of the MOX clones digested with the restrictionenzymes. BamHI, EcoRI/HindIII, HindIII/SalI and PstI/SalI and separatedon 1.5% agarose gels. After hybridization of the blot with a mixture ofboth radioactively labelled probes, the clones 1, 4 and 5 hybridize,while clone 3 does not, as shown for the HindIII/SalI blot in FIG. 8.However, the probes did not hybridize with the EcoRI/HindIII digestedDNA of these clones (result not shown). Since an EcoRI site is presentin the probes, the hybridizing DNA in the clones probably is cut by thisenzyme too. Consequently the hybridization overlap has become too smallto allow the formation of stable hybrids.

Restriction map and sequence analysis

By comparing restriction enzyme digests and by cross-hybridizationexperiments it was concluded that clones 1, 4 and 5 covered identicalstretches of DNA.

In order to definitely establish the nature of this stretch of clonedDNA the insert of clone 4 was analyzed in detail. Hybridization with theamino terminal probe showed that the complete MOX gene (ca. 2 kb) waspresent, including 2 kb sequences upstream and 3.5 kb downstream (FIG.9).

DNA sequence analysis of the smallest EcoRI fragment revealed thenucleotide sequence corresponding to the amino terminus of MOX as wasdetermined by amino acid sequence analysis.

For sequence analysis, several fragments were subcloned in M13mp8/M13mp9or M13mp18/M13mp19 respectively in two orientations, as indicated inFIG. 9. Clones that were smaller than 0.5 kb were sequenced directlyfrom both sides. The larger clones were cut at the unique restrictionsites situated in the middle of the cloned fragment, to allow generationof exonuclease Bal31 digested subclones as described in materials andmethods. Using specific oligonucleotide primers, sequences around therestriction sites used for subcloning and sequences that did not allowan unequivocal sequence determination were sequenced once more, usingthe 5.5 kb BamHI/SacI subclone that covers the whole sequence. Thecomplete nucleotide sequence is given in FIG. 10 and 10A-10H.

The sequence contains an open reading frame of 2046 nucleotides that cancode for a protein of 664 amino acids. The last codon of the openreading frame codes for Phe, which is in agreement with the carboxyterminus of purified MOX. The amino acid composition derived from theDNA sequence encoding this protein, and the amino acid composition ofpurified MOX are virtually identical (Table III). The only importantdifferences involve the serine and threonine residues, which arenotoriously difficult to determine.

The calculated molecular weight of the protein is 74 050 Dalton, whichagrees well with the molecular weight of 74 kd of MOX, as determined onpolyacrylamide/SDS gels.

Codon usage

In Table IV the codon usage for MOX is given. A bias towards the use ofa selective number of codons is evident.

EXAMPLE 2 CONSTRUCTION OF A PLASMID, pUR 3105, BY WHICH THE GENE CODINGFOR NEOMYCIN PHOSPHOTRANSFERASE, THAT CONFERS RESISTANCE AGAINST THEANTIBIOTIC G 418, IS INTEGRATED INTO THE CHROMOSOMAL MOX GENE UNDERREGIE OF THE MOX REGULON.

H. polymorpha cells, transformed with either the plasmids YEP 13, YRP17, pHARS 1 or pHARS 2, were unstable and lost their leu⁺ or ura⁺phenotype already after 10 generations upon growth under non-selectiveconditions. In order to obtain stable transformants and to test the MOXpromoter, a plasmid pUR 3105 is constructed in which the neomycinphosphotransferase gene (NEO^(R)) is brought under direct control of theMOX regulon. The construction is made in such a way that the first ATGof the NEO^(R) gene is coupled to 1.5 kb of the MOX regulon. The cloningof such a large regulon fragment is necessary as shorter fragments, thatdo not contain the -1000 region of the regulon, were less efficient.

The NEO^(R) gene was isolated as a 1.1 kb XmaIII-SalI fragment from thetransposon Tn5, situated from 35 bp downstream of the first ATG up to240 bp downstream of the TGA translational stop codon. To avoid acomplex ligation mixture, first pUR 3101 is constructed (FIGS.11A1-11A2), which is a fusion of the far upstream SalI-XmaIII (position-1510 to position -1128) fragment of the MOX regulon, and the NEO^(R)gene, subcloned on M13mp9. Another plasmid is constructed, pUR 3102, inwhich the 1.5 kb SalI-HgiAI fragment of the MOX gene, that covers nearlythe whole MOX regulon, is ligated to a MOX-NEO^(R) adapter (FIG. 11B)sequence and cloned in M13-mp9. The 1.2 kb XmaIII fragment of thisplasmid is cloned into the XmaIII site of pUR 3101, resulting in pUR3103, which is the exact fusion of the MOX regulon and the NEO^(R) gene(FIG. 11C-11C2). The orientation is checked by cleavage with HgiAI andSalI. From the lambda-MOX-4 clone, a SalI-SacI fragment is subclonedthat reaches from the SalI site, still in the structural MOX gene(position 894), up to the SacI site, far downstream of the structuralMOX gene (position 3259) (see FIG. 9). This M13mp19 subclone is calledpUR 3104. The plasmid pUR 3105 is obtained by the direct ligation of the2.7 kb SalI fragment from pUR 3103 into the SalI site of pUR 3104. Theorientation is tested by cleavage with SmaI and SacI.

After cleavage of this plasmid with HindIII and SacI and thetransformation of this cleaved plasmid to H. polymorpha, G 418-resistantcolonies are found that do not lose their resistance upon growth undernonselective conditions for a large number of generations.

EXAMPLE 3 THE CONSTRUCTION OF pUR 3004, BY WHICH THE GENE CODING FORD-AMINO ACID OXIDASE IS TRANSFERRED TO THE CHROMOSOME OF H. POLYMORPHAUNDER REGIE OF THE MOX-REGULON

D-amino acid oxidase (AAO) is an example of an oxidoreductase for theproduction of which the methylotrophic H. polymorpha is extremelysuited. It might be expected that the enzyme, being an oxidase like MOX,is translocated to the peroxisomes of the yeast that are induced duringgrowth on methanol or a mixture of methanol and a fermentable sugar ascarbon source and D-amino acids as the sole nitrogen source. Under theseconditions the cell will be protected from the H₂ O₂ produced.Alternatively, AAO can be produced without the production of H₂ O₂, whenit is placed under regie of the MOX- or DAS-regulon. The AAO productionwill be induced by the presence of methanol in the medium.

The amino acid sequence of the AAO enzyme has been published (Ronchi etal., 1981) and the complete gene is synthesised, using the phosphitetechnique (Matteuci and Caruthers, 1981). The gene is constructed insuch a way that the optimal codon use for H. polymorpha, as derived fromthe sequence of the MOX gene, is used. Moreover, several uniquerestriction sites are introduced without changing the amino acidsequence, to facilitate subcloning during the synthesis. The DNAsequence is shown in FIG. 12A-12D. The gene is synthesised inoligonucleotides of about 50 nucleotides in length. Oligonucleotides arepurified on 16% polyacrylamide gels. The oligonucleotides that form asubclone are added together in ligase buffer (Maniatis et al., 1982) andheated to 70° C. in a waterbath. The waterbath is slowly cooled to 16°C. and T₄ -ligase is added. After two hours of ligation, the DNA isseparated on a 1.5% agarose gel and the fragment, having the expectedlength, is isolated from the gel. It is subcloned in an M13 mp18 vectorcleaved at the respective restriction sites situated at the end of thefragment. The gene is subcloned in this way in 4 subclones, respectivelySalI-HindIII (position 39-346), HindIII-XmaI (position 346-589),XmaI-KpnI (position 589-721) and KpnI-SalI (position 721-1044). TheSalI-HindIII and HindIII-XmaI subclones and the XmaI-KpnI and Kpn-I-SalIsubclones are ligated together as two SalI-XmaI subclones in SalI-XmaIcleaved M13mp18. These two subclones are ligated into a SalI cleavedM13mp8, resulting in pUR 3001 (FIGS. 12A-12D, 13A1-13A2). The wholesequence is confirmed by the determination of the nucleotide sequenceusing the modified Sanger dideoxy sequencing technique (Biggin et al.,1983).

The construction of the integrative plasmid, containing the AAO gene isshown in FIG. 13A1-13B2, B. The nearly complete AAO gene is placedupstream of the MOX termination region, by insertion of the AAOgene-containing SalI fragment of pUR 3001, in the unique SalI site ofpUR 3104 (see also FIG. 13A1-13A2), resulting in pUR 3002. Theorientation is checked by cleavage with HindIII. The MOX promoter regionis isolated as a 1.4 kb SalI-HgiAI fragment from pUR 3102 (FIG.13A1-13A2). This fragment is subsequently placed upstream of the AAOgene in pUR 3002, by ligation to partially SalI-digested pUR 3002 in thepresence of the HgiAI-SalI MOX-AAO adapter, shown in FIG. 13A1-13A2. Theorientation of the resulting plasmid pUR 3003 is checked again bycleavage with HindIII. This plasmid is integrated into the MOX geneafter cleavage with SacI and transformation to H. polymorpha cells.Transformants are selected by their ability to grow on D-amino acids asnitrogen source in the presence of methanol as inducer.

As the selection of cells containing the AAO gene is not simple, anotherselection marker is introduced. To this end, the S. cerevisiae LEU2 geneis integrated in between the structural AAO gene and the MOX terminater.For this construction, the plasmid pURS 528-03 is used. This plasmid isderived from pURY 528-03 described in European patent application0096910. The construction is shown in FIG. 13C1-13C2. The deletedcarboxy terminal LEU2 gene sequence of pURY 528-03 was replaced by thecomplete carboxy terminal LEU2 gene sequence from pYeleu 10 (Ratzkin andCarbon, 1977) and the E. coli lac-lac regulon was eliminated.Subsequently the HpaI-SalI fragment of pURS 528-03 containing the LEU2gene, is blunt end inserted in the SalI site of pUR 3003, situated inbetween the AAO structural gene and the MOX terminater. The orientationof the resulting plasmid pUR 3004 can be checked by cleavage with SalIand SacI. pUR 3004 integrates in the chromosomal MOX gene of H.polymorpha after transformation of the SacI-cleaved plasmid to a H.polymorpha leu⁻ mutant. Selected leu⁺ transformants are integrated inthe chromosomal MOX gene, together with the AAO gene.

EXAMPLE 4 THE CONSTRUCTION OF pUR 3204, pUR 3205, pUR 3210 and pUR 3211,BY WHICH THE SMALL PEPTIDE HORMONE, THE HUMAN GROWTH RELEASING FACTOR,IS EXPRESSED UNDER REGIE OF THE MOX-REGULON, EITHER BY INTEGRATION INTOTHE CHROMOSOMAL MOX GENE (pUR 3203, pUR 3204), OR BY INTEGRATION INTO AHARS1-CONTAINING PLASMID (pUR 3205) OR BY FUSION TO THE MOX STRUCTURALGENE (pUR 3209, pUR 3210 and pUR 3211)

Human growth hormone releasing factor (HGRF) is a small, 44 amino acidslong, peptide, that activates the secretion of human growth hormone fromthe pituitary glands. HGRF can be used in the diagnosis and treatment ofpituitary dwarfism in man. Since HGRF has been shown to induce growthhormone stimulation in numerous species, HGRF might be used in theveterinary field too, by stimulating growth of animals and increase ofmilk production (Coude et al., 1984). It is difficult to obtain HGRFfrom human sources, but it could very well be produced bybiotechnological processes, once the gene has been cloned andtransferred to an appropriate host organism. Also, as a general exampleof the production of a peptide hormone by H. polymorpha, the gene forHGRF is synthesised in the optimal codon use of H. polymorpha andbrought to expression in several ways.

For the construction of pUR 3204 and pUR 3205, the gene fragment thatcodes for the carboxy terminal part of the protein is synthesised in DNAoligomers of about 50 nucleotides in length and subcloned as aHindIII-SalI fragment in HindIII-SalI cleaved M13mp18, resulting in pUR3201 (FIGS. 14, 15A1-15A2). This HindIII-SalI fragment is subsequentlyinserted upstream of the MOX terminater in HindIII-SalI cleaved pUR 3104(FIG. 15A1-15A2), resulting in pUR 3202. The MOX promoter is inserted infront of the HGRF gene, by insertion of the SalI-HgiAI MOX-promoterfragment from pUR 3102 (FIG. 15A1-15A2) in HindIII cleaved pUR 3202,using a HgiAI-HindIII adapter between the MOX-promoter and the HGRF gene(FIGS. 14, 15A1-15A2). The orientation of the resulting plasmid pUR 3203is checked by cleavage with SalI and HgiAI. pUR 3203 integrates into thechromosomal MOX gene of H. polymorpha after transformation of the SacIcleaved plasmid. Transformants are selected on immunological activity.pUR 3203 is cleaved with SalI, to insert the SalI-HpaI fragment of pURS528-03 (FIG. 15B) that contains the LEU2 gene. The orientation of thisgene in pUR 3204 is checked by cleavage with HindIII and EcoRI. pUR 3204integrates into the chromosomal MOX gene of H. polymorpha aftertransformation of the SacI cleaved plasmid (FIG. 15B) to a leu⁻ H.polymorpha mutant. Selection on on leu⁺ transformants. A plasmid, calledpUR 3205, that replicates autonomously in H. polymorpha and contains theHGRF gene, is obtained by insertion of the EcoRI, partially HindIIIcleaved 4 kb long fragment of pUR 3203, containing the HGRF geneinserted in between the MOX-promoter and terminater, into partiallyHindIII-EcoRI cleaved pHARS1 (FIGS. 2, 15C). The construction of pUR3205 is checked by cleavage with HindIII.

The production of small peptides as HGRF by microorganisms is oftenunstable as a result of enzymic degradation (Itakura et al., 1977).Fusion to a protein like MOX, and subsequent transport to theperoxisomes, could prevent degradation. Therefore, we decided to insertthe HGRF gene into the unique KpnI site at position 1775 (amino acid591, FIGS. 9, 10A-10H ) of the MOX structural gene. The HGRF gene issynthesised again in DNA oligomers of 50 nucleotides in length, but nowas two KpnI-HindIII subclones that are cloned as a complete HGRFstructural gene in M13mp19, cleaved with KpnI (plasmid pUR 3206, FIGS.16, 15D1-15D2). Moreover, the ATG triplet coding for the internalmethionine of HGRF at position 27 (Coude et al., 1984) (position 82 ofthe DNA sequence) is converted into a TGT triplet coding for cysteine.This does not alter the HGRF activity essentially, and facilitates thecleavage of HGRF from the fusion protein by CNBr cleavage (Itakura etal., 1977). From phage lambda MOX-4 (FIG. 9 SphI (position -491)-KpnIfragment is isolated and in serted into SphI-KpnI cleaved M13mp19. Thisresults in pUR 3207. pUR 3206 is cleaved with KpnI and the HGRF gene isinserted into the KpnI site of pUR 3207, resulting in pUR 3208. Theorientation is checked by direct sequence analysis on thesingle-stranded DNA of pUR 3208. Subsequently the downstream part of theMOX gene, from the unique KpnI site up to the SacI site, is isolated asa 1.5 kb fragment from phage lambda MOX-4 and inserted intoSacI--partially KpnI cleaved pUR 3208. The orientation of the resultingplasmid pUR 3209 is checked by digestion with KpnI. pUR 3209 integratesinto the chromosomal MOX gene of H. polymorpha after transformation ofthe SacI, SphI cleaved plasmid. Selection on immunological activity.

This MOX-HGRF fusion gene is inserted into pHARS1 by isolation of thewhole fusion gene from partially HindIII, partially EcoRI cleaved pUR3209, into EcoRI partially HindIII cleaved pHARS1. This results in pUR3210, which replicates in H. polymorpha after transformation (FIG. 15E).Alternatively, the LEU2-containing SalI-HpaI fragment of pURS 528-03 isinserted into the blunt-ended KpnI site of the HGRF gene, located at thecarboxy terminus of the encoded protein, after partial KpnI cleavage ofpUR 3209. The resulting plasmid pUR 3211 integrates into the chromosomalMOX gene of H. polymorpha, after transformation of the SacI, SphIcleaved plasmid (FIG. 15F).

Discussion

From the length of the open reading frame, from the similarity in theamino acid composition of purified MOX and the DNA derived proteinsequence and from the identical 30 N-terminal amino acids, it isconcluded that the complete gene for MOX from the yeast Hansenulapolymorpha has been cloned. Its calculated molecular weight agrees wellwith the molecular weight determined on SDS polyacrylamide gels. Apartfrom the coding sequence, more than 1200 bp has been sequenced from boththe 5'- and the 3'-non-coding regions, reaching from the SalI siteupstream of the coding sequence, up to the SacI site downstream. Thegene appears not to be interrupted with intervening sequences.

The protein is not transcribed in the form of a precursor. Based on thedetermination of the molecular weight, N-terminal signal sequences couldnot be detected in earlier studies of Roa and Blobel (1983) orRoggenkamp et al. (1984) as well. In similar studies, it was suggestedthat also the rat liver peroxisomal enzymes uricase (Goldman and Blobel,1978) and catalase (Goldman and Blobel, 1978; Robbi and Lazarow, 1978)do not contain a cleavable N-terminal signal peptide. However, asdiscussed by these authors, proteolytic degradation could possiblyexplain the lack of the detection of such a signal sequence.

Our sequence results definitely prove that for translocation of thisprotein to the peroxisome, a cleavable N-terminal signal sequence is notrequired. Such a translocation signal may well be situated in theinternal sequence of the mature protein, as is the case for ovalbumine(Lingappa et al., 1979). Inspection of the protein sequence reveals theamino acid sequence Gly X Gly Y Z Gly (amino acids 13-18), which ischaracteristic for FAD-(flavin adenine dinucleotide)-containing enzymes(Ronchi et al., 1981).

The isolation of the MOX gene described above gives a way how todetermine the DNA sequence coding for MOX and the amino acid sequence ofthe MOX enzyme.

Similarly, the DNA sequences and amino acid sequences belonging to otheroxidase-enzymes can be isolated and determined. The knowledge of the MOXgene sequence can be used to facilitate the isolation of genes codingfor alcohol oxidases or even other oxidases. By comparing the propertiesand the structure of enzymes one can probably establish structurefunction and activity relationships. One can also apply methods assite-directed mutagenesis, or shortening or lengthening of the proteincoding sequences, modifying the corresponding polypeptides, to selectoxidase-enzymes with improved properties, e.g. with increased alkalistability, improved production, or oxidase-enzymes which need asubstrate which is more compatible with detergent products.

Besides the isolation and characterization of the structural gene forMOX from the yeast H. polymorpha, also the isolation andcharacterization of the structural gene for DHAS from the yeast H.polymorpha has been carried out in a similar way.

The DNA sequence of DAS is given in FIG. 17A-17H. A restriction map isgiven in FIG. 18A-18B. The amino acid composition calculated from theDNA sequence of DAS appeared to be in agreement with the amino acidcomposition determined after hydrolysis of purified DHAS. The DHASenzyme catalyses the synthesis of dihydroxyacetone from formaldehyde andxylulose monophosphate. This reaction plays a crucial role in themethanol-assimiliation process (cf. Veenhuis et al., 1983).

As described before, the synthesis of MOX and DHAS is subject to glucoserepression. It has now been found that higher levels of MOX are reachedwhen using glucose/methanol mixtures as substrates instead of 0.5% (v/v)methanol. Under the former conditions up to 30% of the cellular proteinconsists of MOX, compared with up to 20% under the latter conditions.

It was considered that in the regulons of MOX and DAS sequences mustexist that play a decisive role in the regulation ofrepression/derepression by glucose or of the induction by methanol. Somehomology therefore might be expected.

A striking homology of the "TATA-boxes" has been found, both having thesequence CTATAAATA. No other homologies in the near upstream region ofthe MOX and DAS regulons have been found. Unexpectedly, a detailed studyof both regulons has shown a remarkable homology of the regulons for MOXand DAS in the region about 1000 bp upstream of the translationinitiation codon. A practically complete consecutive region of 65 bp inthe regulon of MOX is homologous to a 139 bp region in the DAS regulon,interspersed by several non-homologous regions (see FIG. 19). A similarhomology is not found in any other region of both genes, that are over 4kb in length including their upstream and downstream sequences. It issuggested that these homologous sequences play a role in the regulationof both genes by glucose and methanol. Transformation studies withvectors containing as regulon the first 500 bp upstream of the ATG ofthe structural gene of MOX, showed that this shortened MOX-regulon gaverise to a relatively low expression of the indicator genebeta-lactamase. Indicator genes are genes which provide the yeast withproperties that can be scored easily, e.g. the gene for neomycinphosphotransferase giving resistance to the antibiotic G 418 (cf. Watsonet al., 1983) or an auxotrophic marker such as leucine.

The fact that the far upstream homologous regions in the MOX and DASgenes have different interruptions and the fact that DAS is repressed at0.1% glucose and MOX is not, suggest that these homologous regions areof importance to the repression-derepression by glucose and/or theinduction of the expression in the presence of methanol. This assumptionhas been found correct indeed, and the presence or absence of thesehomologous regions can therefore be important for specific applications.For example, if the -1052 to -987 region of the MOX gene or the -1076 to-937 region of the DAS gene is important for the induction of MOX or DASby methanol, the presence of these regions is required for theexpression of MOX or DAS and/or for the induction of other enzymes bymethanol. Another example might be the removal of the regions to avoidrepression by glucose, which is needed for the expression of genescoding for proteins other than MOX and DHAS under influence of the MOXand/or DAS regulatory regions with glucose as a carbon source.

Thus one aspect of the present invention relates to the isolation andcomplete characterization of the structural genes coding for MOX andDHAS from the yeast H. polymorpha. It further relates to the isolationand complete characterization of the DNA sequences that regulate thebiosynthesis of MOX and DHAS in H. polymorpha, notably the regulons andterminaters.

Moreover, it relates to combinations of genes coding for alcohol oxidaseor other oxidases originating from H. polymorpha strains other than H.polymorpha CBS 4732, or Hansenula species other than H. polymorpha, oryeast genera other than Hansenula, or moulds, or higher eukaryotes, withthe powerful regulon and terminater of the MOX gene from H. polymorphaCBS 4732. These combinations may be located on vectors carrying amongstothers an autonomously replicating sequence originating from H.polymorpha or related species or minichromosomes containing centromeres,and optionally selection marker(s) and telomeres. These combinations mayalso be integrated in the chromosomal DNA of H. polymorpha.

Furthermore it relates to combinations of the powerful regulon or partsof it and terminaters of the MOX and/or DAS and--by site-directedmutagenesis or other methods--changed structural genes coding foralcohol oxidase or another oxidase. These changed structural genes maybe located on episomal vectors, in minichromosomes or integrated in thechromosomes of H. polymorpha, H. wingeii, H. anomala, and S. cerevisiaeor in other yeasts.

Besides this, the present invention relates to combinations of theregulon and terminater of the MOX and/or DAS gene of H. polymorpha withstructural genes coding for other proteins than oxidases.

A very important and preferred embodiment of the invention is a processfor preparing a polypeptide, such as a protein or an enzyme, byculturing a microorganism under suitable conditions, optionallyconcentrating the polypeptide and collecting same in a manner known perse, characterized in that a microorganism is used that has been obtainedby recombinant DNA technology and caries a structural gene coding forthe polypeptide concerned, the expression of which is under the controlof a regulon, comprising a promoter and at least either the -1052 to-987 region of the MOX gene of Hansenula polymorpha CBS 4732, or the-1076 to -937 region of the DAS gene of Hansenula polymorpha CBS 4732,or a corresponding region of other methylotrophic moulds or yeasts, oran effective modification of any of these regions.

Surprisingly, it has been observed by the present inventors that theregions concerned, which are shown in FIG. 20 and are referred to hereinas the -1000 regions of the MOX and DAS genes, are of crucial importancefor the expression of the structural gene concerned. Experimentsperformed with recombinants containing the MOX regulon from which thisregion was eliminated showed a low level of expression. Therefore, useof a regulon comprising such -1000 region, or an effective modificationthereof, i.e. any modification which does not result in a significantmutilation of the function of said region, makes it possible to realizeproduction of a relatively high amount of the desired polypeptide.

A preferred embodiment of this process according to the invention ischaracterized in that the structural gene concerned has been providedwith one or more DNA sequences coding for amino acid sequences involvedin the translocation of the gene product into the peroxisomes orequivalent microbodies of the microbial host. Translocation of theproduced polypeptide into the peroxisomes or equivalent microbodiesimproves their stability, which results in a higher yield. For certainkinds of polypeptides, in particular oxidases, such translocation isimperative for survival of the microbial host, i.e. to protect the hostagainst the toxic effects of the hydrogen peroxide produced when themicrobial host cells are growing on the substrate of the oxidase. If theoxidase concerned does not contain addressing signals which arefunctional in the microbial host used in the production process, oneshould provide the structural gene with sequences coding for hostspecific addressing signals, for example by adding such sequences or bysubstituting these for the original addressing sequences of the gene.Production of a fused polypeptide, in which the fusion partner carriessuitable addressing signals, is another possibility. In casemethylotrophic yeasts are used in the production process, it ispreferred that the DNA sequences consist of the MOX gene or those partsthereof which are responsible for MOX translocation into the peroxisomesor microbodies.

Finally, this aspect of the present invention is related to thesynthesis of MOX originating from H. polymorpha in other yeasts.

Some microorganisms with the potential of producing alcohol oxidases aresummarized below.

    ______________________________________                                        Yeasts producing alcohol oxidases                                             (Taxonomic division according to Lee and Komagata, 1980)                      Group 1         Candida boidinii                                              Group 2a        Hansenula philodendra                                                         Pichia lindnerii                                                              Torulopsis nemodendra                                                         Torulopsis pinus                                                              Torulopsis sonorensis                                         Group 2b        Candida cariosilignicola                                                      Hansenula glucozyma                                                           Hansenula henricii                                                            Hansenula minuta                                                              Hansenula nonfermentans                                                       Hansenula polymorpha                                                          Hansenula wickerhamii                                                         Pichia pinus                                                                  Pichia trehalophila                                           Group 2c        Candida succiphila                                                            Torulopsis nitratophila                                       Group 3         Pichia cellobiosa                                             Group 4         Hansenula capsulata                                                           Pichia pastoris                                                               Torulopsis molischiana                                        Moulds producing alcohol oxidases:                                                          Lenzites trabea                                                               Polyporus versicolor                                                          Polyporus obtusus                                                             Poria contigua                                                  ______________________________________                                    

Among the oxidases other than alcohol oxidases, the most interestingare:

glycerol oxidase,

aldehyde oxidase,

amine oxidase,

aryl-alcohol oxidase,

amino acid oxidase,

glucose oxidase,

galactose oxidase,

sorbose oxidase,

uric acid oxidase,

chloroperoxidase, and

xanthine oxidase.

Combinations of the powerful regulons and terminaters of the MOX and DASgenes from H. polymorpha and structural genes for oxidases may becombined with one or more DNA sequences that enable replication of thestructural gene in a particular host organism or group of hostorganisms, for example autonomously replicating sequences or centromers(and telomers) originating from H. polymorpha, to suitable vectors thatmay be transferred into H. polymorpha and related yeasts or othermicroorganisms.

H. polymorpha mutants LEU-1 and LR9, mentioned on page 12 of thisspecification, were deposited at the Centraalbureau voorSchimmelcultures at Delft on 15th Jul., 1985, under numbers CBS 7171 andCBS 7172, respectively.

The above description is followed by a list of references, claims,Tables, Legends to Figures and Figures.

                  TABLE I                                                         ______________________________________                                        Activities of orotidine 5'-phosphate decarboxylase and                        orotidine 5'-phosphate pyrophosphorylase in H. polymorpha                     mutants requiring uracil for growth.                                                       Activity (%).sup.a                                                                  Orotidine 5'-                                                                             Orotidine 5-                                   Strain/  Reversion phosphate   phosphate                                      Genotype rate      decarboxylase                                                                             pyrophosphorylase                              ______________________________________                                        Wild type                                                                              --        100         100                                            LR 9/odc1                                                                              <2 × 10.sup.9                                                                     <1          106                                            MR 7/odc1                                                                               6 × 10.sup.7                                                                     <1          71                                             NM 8/odc1                                                                               3 × 10.sup.8                                                                     <1          105                                            CLK 55/opp1                                                                            n.e..sup.b                                                                              90          <1                                             CLK 68/opp1                                                                            n.e.      82          <1                                             YNN 27/ura3                                                                            n.e.      0           n.e.                                           ______________________________________                                         Strains were grown in YEPD until late exponential phase. Extraction of        cells was performed with glass beads using a Braun homogenizer. Protein       was estimated by the optical density at 280 nm.                               .sup.a) Expressed as the percentage of wild type activity.                    .sup.b) Not estimated.                                                   

                  TABLE II                                                        ______________________________________                                        Transformation of uracil-requiring mutants of H. polymorpha                                                       Status of                                                  Transformation                                                                            Stability.sup.b                                                                      transformed                               Strain  Plasmid  frequency.sup.a                                                                           (%)    DNA                                       ______________________________________                                        LR 9    YRP17    2.2 × 10.sup.2                                                                      <1     Autonomous                                                                    replication                               LR 9    pHARS1   1.5 × 10.sup.3                                                                      2      Autonomous                                                                    replication                               LR 9    pHARS2   4.6 × 10.sup.2                                                                      1.5    Autonomous                                                                    replication                               LR 9    YIP5     3 (38).sup.c                                                                              105    Integration                               LR 9    pRB58    0           --     --                                        LR 9    pHH85    0           --     --                                        YNN 27  YIP5     0           --     --                                        ______________________________________                                         .sup.a) Expressed as total number per μg of DNA. Intact cells treated      with polyethyleneglycol were used for transformation as described in          Materials and Methods.                                                        .sup.b) Expressed as the percentage of remaining uracil prototrophs after     growth on YEPD for ten generations.                                           .sup.c) Number in parentheses indicates the amount of minicolonies            containing free plasmid YIP5.                                            

                  TABLE III                                                       ______________________________________                                        Amino acid composition of MOX                                                 Amino Acid   DNA sequence                                                                             Hydrolysate.sup.a)                                    ______________________________________                                        PHE          31                     32                                        LEU          47                     49                                        ILE          34                     34                                        MET          12                     11                                        VAL          42                     43                                        SER          43                     33.sup.a)                                 PRO          43                     42                                        THR          44                     38                                        ALA          47                     50                                        TYR          27                     27                                        HIS          19                     21                                         GLN          13                                                                                                  51                                        GLU          36                                                                ASN          32                                                                                                  84                                        ASP          50                                                               LYS          35                     38                                        CYS          13                     12                                        TRP          10                     --.sup.b)                                 ARG          36                     36                                        GLY          50                     53                                        ______________________________________                                         .sup.a) Hydrolysis was performed for 24 h.                                    .sup.b) Not determined.                                                  

                  TABLE IV                                                        ______________________________________                                        Comparison of preferred codon usage in S. cerevisiae,                         H. polymorpha and E. coli                                                                   Hansenula                                                       Saccharomyces MOX            E. coli                                          ______________________________________                                        ALA   GCU, GCC    GCC            GCC not used,                                                                 no clear pref.                               SER   UCU, UCC    UCC, UCG       UCU, UCC                                     THR   ACU, ACC    ACC            ACU, ACC                                     VAL   GUU, GUC    GUA not used,  GUU, GUA                                                       no clear pref.                                              ILE   AUU, AUC    AUC, AUU       AUC                                          ASP   GAC         GAC            GAC                                          PHE   UUC         UUC            UUC                                          TYR   UAC         UAC            UAC                                          CYS   UGU         no clear pref. no clear pref.                               ASN   AAC         AAC            AAC                                          HIS   CAC         CAC            CAC                                          GLU   GAA         GAG            GAA                                          GLY   GGU         GGC practically                                                                              GGU, GGC                                                       not used, no clear pref.                                    GLN   CAA         CAG            CAG                                          LYS   AAG         AAG            AAA                                          PRO   CCA         CCU, CCA       CCG                                          LEU   UUG         CUG, CUC       CUG                                          ARG   AGA         AGA            CGU                                          ______________________________________                                    

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Legends to Figures

FIG. 1. The exonuclease Bal31 digestion strategy used in sequencingspecific MOX subclones. The fragment X-Y subcloned in M13mp-8 or -9, -18or -19 is cut at the unique restriction site Z. The DNA molecule issubjected to a time-dependent exonuclease Bal31 digestion. The DNAfragment situated near the M13 sequencing primer is removed usingrestriction enzyme Y; ends are made blunt end by incubation with T₄ -DNApolymerase and then ligated intramolecularly. Phage plaques are pickedup after transformation and the fragment is sequenced from site Z in thedirection of site X. Using the M13 derivative with a reversed multiplecloning site, the fragment is sequenced from site Z in the direction ofsite X.

FIG. 2. Alignment of pHARS plasmids derived by insertion of HARSfragments into the single SalI site of YIp5.

FIG. 3. Estimation of copy number by Southern hybridization of H.polymorpha transformants. An aliquot of 8 and 16 μl of each probe waselectrophoresed. Lane 1, phage lambda DNA digested with HindIII andEcoRI. Lanes 2,3 transformant of K. lactis containing two copies ofintegrated plasmid, digested with HindIII (M. Reynen, K. Breunig and C.P. Hollenberg, unpublished); lanes 4-7, YNN 27, transformed with pRB58(4-5) and YRP17 (6-7) digested with EcoRI respectively; lanes 8,9, LR9transformed with YRP17 digested with EcoRI; lanes 10,11, LR9 transformedwith pHARS2 digested with HindIII; lanes 12,13, LR9 transformed withpHARS1 digested with EcoRI.

FIG. 4. Autoradiogram of Southern blots of DNA from H. polymorpha mutantLR9 transformed by integration of plasmid YIp5. Lane 1, phage lambdaDNA, digested both with HindIII and EcoRI; lane 2, pHARS-1, undigested;lanes 3-5 and lanes 6,7 show DNA from 2 different transformants. Lane 3,undigested; lane 4, digested with EcoRI; lane 5, digested with PvuII;lane 6, digested with EcoRI; lane 7, digested with PvuII; lane 8,plasmid YIp5, digested with EcoRI. Nick-translated YIp5 was used as ahybridization probe.

FIG. 5. Electrophoresis of ³² p-labelled RNA from Hansenula polymorpha,purified once (lane A) or twice (lane B) on oligo(dT)cellulose.Electrophoresis was performed on a denaturing 7M urea 2.5%polyacrylamide gel. The position of the yeast rRNA's and theirrespective molecular weights are indicated by 18 S and 25 S. The 2.3 kbband, that can be seen in lane B, was converted into a cDNA probe whichwas subsequently used to isolate MOX and DHAS clones from the Hansenulapolymorpha clone bank.

FIG. 6. ³⁵ S-labelled proteins obtained after in vitro translation ofmethanol derepressed, Hansenula polymorpha mRNA with a rabbitreticulocyte lysate. Either 2 microliters of the total lysate (lane A)or an immuno-precipitate of the remaining 18 microliters using a MOXspecific antiserum (lane B) were separated on an 11.5%SDS-polyacrylamide gel. A mixture of proteins with known molecularweights was used as markers.

FIG. 7. The N-terminal sequence of purified MOX, as determined on aBeckman sequenator. The two probes that could be derived from thesequence Pro-Asp-Gln-Phe-Asp, using Saccharomyces preferred codons, areindicated.

FIG. 8A-8B. Hybridization of a DBM blot of HindIII/SalI cut MOX clones.The DNA was separated on a 1.5% agarose gel (FIG. 8A) and the blot washybridized to a mixture of both MOX-derived synthetic DNA probes (FIG.7). Only one band of clones 1, 4 and 5 hybridize (FIG. 8B), indicated byan arrow in FIG. 8A. Lane M: molecular weight markers as indicated. LaneA, B, C and D: clones 1, 3, 4 and 5, respectively. Lane E: lambda L47.1.

FIG. 9. Restriction map for MOX clone 4. Only relevant restriction sitesare indicated that have been used for subcloning and sequencing of theMOX gene. The open reading frame, containing the structural MOXsequence, and the M13 subclones made are depicted. Restriction sitesused are: B=BamHI, E_(I) =EcoRI, E_(V) =EcoRV, P=PstI, Sl=SalI, Sc=SacI,St=StuI, H=HindIII, Sp=SphI, K=KpnI, Hg=HgiAI and X=XmaI.

FIG. 10A-10H. The nucleotide sequence of the MOX structural gene and its5'- and 3'-flanking sequence.

FIG. 11A, A2 and 11C1, C2. The construction of plasmid pUR 3105 by whichthe neomycin phosphotransferase gene integrates into the chromosomal MOXgene of H. polymorpha.

FIG. 11B. Promoter MOX-neomycin phosphotransferase adapter fragments.

FIG. 12A-12D. The DNA sequence of the AAO gene, derived from thepublished amino acid sequence. The gene is synthesised in the optimalcodon use for H. polymorpha in oligonucleotides of about 50 nucleotideslong. Restriction sites, used for subcloning are indicated. TheHgiAI-SalI fragment forms the adapter between the structural AAO geneand the MOX promoter. The translational start codon (met) and stop codon(***) are indicated. The structural sequence is numbered from 1 to 1044,while the MOX promoter is numbered from -34 to -1.

FIG. 13A1 -13A2. The construction of pUR 3003, by which the AAO geneintegrates into the chromosomal MOX gene of H. polymorpha. Selection onactivity of the AAO gene.

FIG. 13B1-13B2. The construction of pUR 3004, by which the AAO geneintegrates into the chromosomal MOX gene of a H. polymorpha leu⁻derivative. Selection on leu⁺.

FIG. 13C1-13C2. The construction of pURS 528-03. Owing to the removal ofthe pCR1 sequence and the double lac UV5 promoter, this plasmid is about2.2 kb shorter than pURY 528-03.

FIG. 14. The DNA sequence of the HGRF gene, derived from the publishedamino acid sequence. The gene is synthesised in the optimal codon usefor H. polymorpha in oligonucleotides of about 50 nucleotides long.HgiAI, HindIII and SalI sites are used for subcloning. The HgiAI-HindIIIfragment forms the adapter between the structural HGRF gene and the MOXpromoter. The translational start codon (met) and stop codon ***) areindicated. The structural sequence is numbered from 1 to 140, while theMOX promoter is numbered from -34 to -1.

FIG. 15A1-15A2. The construction of pUR 3203, by which the gene codingfor HGRF integrates into the chromosomal MOX gene of H. polymorpha.Selection on immunological activity of HGRF.

FIG. 15B. The construction of pUR 3204, by which the gene coding forHGRF integrates into the chromosomal MOX gene of a H. polymorpha leu⁻derivative. Selection on leu⁺.

FIG. 15C. The construction of pUR 3205, by which the gene coding forHGRF is inserted into a HARS-1-containing plasmid, which replicatesautonomously in H. polymorpha. Selection by transformation of a ura⁻mutant.

FIG. 15D1-15D2. The construction of pUR 3209, by which the gene codingfor HGRF integrates into the chromosomal MOX gene of H. polymorpha,fused to the structural MOX gene. HGRF is cleaved from the fusionprotein by CNBr cleavage. Selection on immunological activity of HGRF.

FIG. 15E. The construction of pUR 3210, by which the gene coding forHGRF is inserted into a HARS-1-containing plasmid, fused to thestructural MOX gene. Selection as in FIG. 15C.

FIG. 15F. The construction of pUR 3211, by which the gene coding forHGRF integrates into the chromosomal MOX gene of a H. polymorpha leu⁻derivative, fused to the structural MOX gene. Selection on leu⁺.

FIG. 16. The DNA sequence of the HGRF gene, derived from the publishedamino acid sequence. The gene is synthesised as mentioned in FIG. 14,but constructed in such a way that it could be inserted into the uniqueKpnI site of the structural MOX gene. Therefore it was equipped withKpnI sites on both sides of the gene, and KpnI-HindIII fragments wereused for subcloning. Synthesis will be as a fusion product to the MOXenzyme. The internal met (ATG) at position 82 is converted into a cys(TGT). Translational start (met) and stop (***) codons are indicated.

FIG. 17A-17H. The nucleotide sequence of the DAS structural gene and its5'- and 3'-flanking sequence.

FIG. 18A-18B. Restriction map for the DAS-lambda clone. Only relevantrestriction sites are indicated that have been used for subcloning andsequencing of the MOX gene. The open reading frame, containing thestructural DAS sequence, and the M13 subclones made, are depicted.

FIG. 19. Identical sequences in -1000 region of DAS and MOX genes.

We claim:
 1. Process for preparing a polypeptide comprising culturing aHansenula polymorpha transformed with a structural gene coding for thepolypeptide under conditions such that said structural gene is expressedand said polypeptide is thereby produced, wherein said structural geneis operably linked to a region comprising a promoter selected from thegroup consisting of at least the -1 to about -1500 region of theHansenula polymorpha MOX gene and the -1 to -2125 region of theHansenula polymorpha DAS gene, said structural gene being other than anative MOX or DAS gene.
 2. Process for preparing a polypeptidecomprising culturing a fungus of a genera selected from the groupconsisting of Hansenula and Saccharomyces transformed with a structuralgene coding for the polypeptide under conditions such that saidstructural gene is expressed and said polypeptide is thereby produced,wherein said structural gene is operably linked to a region comprising apromoter selected from the group consisting of at least the -1 to about-1500 region of the Hansenula polymorpha MOX gene and the -1 to -2125region of the Hansenula polymorpha DAS gene, said structural gene beingother than a native MOX or DAS gene.
 3. The process of claim 1 whereinsaid MOX gene is given in FIGS. 10A and 10B.
 4. The process of claim 2wherein said MOX gene is given in FIG.
 10. 5. The process of claim 1wherein said DAS gene is given in FIGS. 17A, 17B and 17C.
 6. The processof claim 2 wherein said DAS gene is given in FIG.
 17. 7. The process ofclaim 1 wherein said structural gene is linked to a terminator selectedfrom the group consisting of the terminator designated 1993 to about3260 in FIGS. 10A and 10B and the terminator designated 2110 to about2350 in FIGS. 17A, 17B and 17C.
 8. The process of claim 2 wherein saidstructural gene is linked to a terminator selected from the groupconsisting of the terminator designated 1993 to about 3260 in FIG. 10and the terminator designated 2110 to about 2350 in FIG 17.